We investigated the efficiency of target-enriched long-read sequencing (TELSeq) for detecting antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) within complex matrices. We aimed to overcome limitations associated with traditional antimicrobial resistance (AMR) detection methods, including short-read shotgun metagenomics, which can lack sensitivity, specificity, and the ability to provide detailed genomic context. By combining biotinylated probe-based enrichment with long-read sequencing, we facilitated the amplification and sequencing of ARGs, eliminating the need for bioinformatic reconstruction. Our experimental design included replicates of human fecal microbiota transplant material, bovine feces, pristine prairie soil, and a mock human gut microbial community, allowing us to examine variables including genomic DNA input and probe set composition. Our findings demonstrated that TELSeq markedly improves the detection rates of ARGs and MGEs compared to traditional sequencing methods, underlining its potential for accurate AMR monitoring. A key insight from our research is the importance of incorporating mobilome profiles to better predict the transferability of ARGs within microbial communities, prompting a recommendation for the use of combined ARG–MGE probe sets for future studies. We also reveal limitations for ARG detection from low-input workflows, and describe the next steps for ongoing protocol refinement to minimize technical variability and expand utility in clinical and public health settings. This effort is part of our broader commitment to advancing methodologies that address the global challenge of AMR.

中文翻译：

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影响抵抗组和移动组的靶标富集长读长测序的因素


我们研究了靶向富集长读长测序 （TELSeq） 检测复杂基质中抗菌素耐药基因 （ARG） 和移动遗传元件 （MGE） 的效率。我们旨在克服与传统抗菌素耐药性 （AMR） 检测方法相关的限制，包括短读长鸟枪法宏基因组学，该方法可能缺乏敏感性、特异性和提供详细基因组背景的能力。通过将基于生物素化探针的富集与长读长测序相结合，我们促进了 ARG 的扩增和测序，无需生物信息学重建。我们的实验设计包括人类粪便微生物群移植材料、牛粪、原始草原土壤和模拟人类肠道微生物群落的重复，使我们能够检查包括基因组 DNA 输入和探针组组成在内的变量。我们的研究结果表明，与传统测序方法相比，TELSeq 显著提高了 ARGs 和 MGEs 的检出率，强调了其准确监测 AMR 的潜力。我们研究的一个关键见解是结合移动组谱以更好地预测 ARGs 在微生物群落内的可转移性的重要性，这促使建议在未来的研究中使用组合 ARG-MGE 探针组。我们还揭示了低输入工作流程中 ARG 检测的局限性，并描述了持续改进方案的后续步骤，以最大限度地减少技术差异并扩大在临床和公共卫生环境中的实用性。这项工作是我们推进应对 AMR 全球挑战的方法的更广泛承诺的一部分。